High specificity of the cDNA-RNase assay to detect accurate splicing in vitro

e previously developed a splicing assay (Keohavong et al., 1982) that we designated as a cDNA-RNase assay to analyze the ligation reaction between exons of premessenger RNA during in vivo or in vitro splicing. It was important to determine the specificity of this splicing assay, since the accuracy of in vitro splicing must always be demonstrated clearly. To do this, we constructed DNA probes derived from adenovirus E1A cDNA carrying deletions or insertions of 2-6 bases. After hybridizing them to the wild-type mRNA, the ability of single-strand-specific RNases to detect small mismatches of the RNA-DNA hybrids was examined. The demonstration that an imprecision in the splicing reaction of as little as 2 nucleotides can be detected with an efficiency of 99% indicates the high specificity of the splicing assay and its usefulness for the verification of accurate splicing in in vitro systems.